WO2010112194A1 - Antigen-binding polypeptides and multispecific antibodies comprising them - Google Patents

Antigen-binding polypeptides and multispecific antibodies comprising them Download PDF

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WO2010112194A1
WO2010112194A1 PCT/EP2010/002008 EP2010002008W WO2010112194A1 WO 2010112194 A1 WO2010112194 A1 WO 2010112194A1 EP 2010002008 W EP2010002008 W EP 2010002008W WO 2010112194 A1 WO2010112194 A1 WO 2010112194A1
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antibody
linker
vh
vl
cl
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PCT/EP2010/002008
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French (fr)
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Christian Klein
Wolfgang Schaefer
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F. Hoffmann-La Roche Ag
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55Fab or Fab'
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622Single chain antibody (scFv)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide

Abstract

The present invention relates to relates to antigen-binding polypetides comprising a) VH-CL-linker-VL-CH1 or b) VL-CH1-linker-VH-CL and especially to multispecific antibodies comprising such polypetides, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

Description

Antigen-binding polypeptides and multispecific antibodies comprising them

The present invention relates to relates to antigen-binding polypetides comprising a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL and especially to multispecific antibodies comprising such polypetides., methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

Background of the Invention

A wide variety of multispecific recombinant antibody formats have been developed in the recent past, e.g. tetravalent bispecific antibodies by fusion of, e.g., an IgG antibody format and single chain domains (see e.g. Coloma, M.J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S.L., Nature Biotech

25 (2007) 1233-1234).

Also several other new formats wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- or tetrabodies, minibodies, several single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed (Holliger, P., et al., Nature Biotech 23 (2005)

1126-1136; Fischer, N., Leger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal of Immunological Methods 318 (2007) 65-74; Wu, C, et al., Nature Biotech. 25 (2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer, N., Leger, O., Pathobiology 74 (2007) 3-14). It has to be kept in mind that one may want to retain effector functions, such as e.g. complement- dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC), which are mediated through the Fc receptor binding, by maintaining a high degree of similarity to naturally occurring antibodies.

In WO 2007/024715 are reported dual variable domain immunoglobulins as engineered multivalent and multispecific binding proteins. A process for the preparation of biologically active antibody dimers is reported in US 6,897,044. Multivalent Fv antibody construct having at least four variable domains which are linked with each over via peptide linkers are reported in US 7,129,330. Dimeric and multimeric antigen binding structures are reported in US 2005/0079170. Tri- or tetra-valent monospecific antigen-binding protein comprising three or four Fab fragments bound to each other covalently by a connecting structure, which protein is not a natural immunoglobulin are reported in US 6,511,663. In WO 2006/020258 tetravalent bispecifϊc antibodies are reported that can be efficiently expressed in prokaryotic and eukaryotic cells, and are useful in therapeutic and diagnostic methods. A method of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers is reported in US 2005/0163782. Bispecific tetravalent receptors are reported in US 5,959,083. Engineered antibodies with three or more functional antigen binding sites are reported in WO 2001/077342.

Multispecific and multivalent antigen-binding polypeptides are reported in WO 1997/001580. WO 1992/004053 reports homoconjugates, typically prepared from monoclonal antibodies of the IgG class which bind to the same antigenic determinant are covalently linked by synthetic cross-linking. Oligomeric monoclonal antibodies with high avidity for antigen are reported in WO 1991/06305 whereby the oligomers, typically of the IgG class, are secreted having two or more immunoglobulin monomers associated together to form tetravalent or hexavalent IgG molecules. Sheep-derived antibodies and engineered antibody constructs are reported in US 6,350,860, which can be used to treat diseases wherein interferon gamma activity is pathogenic. In US 2005/0100543 are reported targetable constructs that are multivalent carriers of bi-specific antibodies, i.e., each molecule of a targetable construct can serve as a carrier of two or more bi-specific antibodies. Genetically engineered bispecific tetravalent antibodies are reported in WO 1995/009917. In WO 2007/109254 stabilized binding molecules that consist of or comprise a stabilized scFv are reported.

Speck, R.R., et al., Hybridoma 16 (1997) 243-248, relates to Biodistribution of Anti-Breast Mucin HuBrE3 -Derived Inverted Fabs (IFabs) in Nude Mice Carrying MX-I Xenografts and inter alia to IFabs as multimers of VH-CHl -linker- VK-CK monomers. Inoue, Y., et al ; Appl Microbiol Biotechnol 48 (1997) 487-492 relate to the efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis. Hust, M., et al., BMC Biotechnology 7 (2007) 14, relates to single chain Fab (scFab) fragments which should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Jordan, E., et al, Microbial Cell Factories 6 (2007) 38 relates to the production of single chain Fab (scFab) fragments in Bacillus megaterium. US 2007/0274985 relates to antibodies comprising single chain Fab (scFab) fragments.

Summary of the Invention

A first aspect of the current invention is a polypetide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHl), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker are connected in one of the following orders from N-terminal to C-terminal direction: a) VH-CL-linker- VL-CHl and b) VL-CHl -linker- VH-CL; wherein VH and VL form together an antigen-binding site which binds specifically to an antigen and wherein said linker is a polypeptide of at least 30 amino acids.

Another aspect of the current invention is a mono- or multispecific antibody comprising i) two or more of said polypetides a) VH-CL-linker- VL-CHl or b) VL- CHl -linker- VH-CL; binding to one or more antigens; and wherein said polypeptides are fused via a peptide connector(s) with each other.

Another aspect of the current invention is a multispecific antibody comprising i) one or more of said polypetides a) VH-CL-linker- VL-CHl or b) VL- CHl -linker- VH-CL; binding to one or more antigens; and ii) a full length antibody specifically binding to a further antigen and consisting of two antibody heavy chains and two antibody light chains; and wherein said polypeptides under under i) are fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody.

Another aspect of the current invention is said multispecific antibody comprising i) one to four of said polypetides a) VH-CL-linker- VL-CHl or b) VL- CHl -linker- VH-CL; binding to one or more antigens; and ii) a full length antibody specifically binding to a further antigen and - A -

consisting of two antibody heavy chains and two antibody light chains; and wherein said polypeptides under under i) are fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody.

Another aspect of the current invention is said multispecific antibody comprising i) one of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL; binding to one antigen; and ii) a full length antibody specifically binding to one further antigen and consisting of two antibody heavy chains and two antibody light chains; and wherein said polypeptide under under i) is fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody.

Another aspect of the current invention is said multispecific antibody comprising i) two of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL; binding to one antigen; and ii) a full length antibody specifically binding to one further antigen and consisting of two antibody heavy chains and two antibody light chains; and wherein said polypeptides under under i) are fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody.

Preferably the said multispecific antibody comprises one or two polypetides a) VH- CL-linker- VL-CHl or b) VL-CHl -linker- VH-C L, binding to the same antigen (bispecific antibody).

Preferably the said multispecific antibody comprises two identical polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL, binding to the same antigen (bispecific antibody).

Preferably the said multispecific antibody comprise two polypetides a) VH-CL- linker- VL-CHl or b) VL-CHl -linker- VH-CL binding to a first antigen and a second antigen (trispecific antibody). A further aspect of the invention is a nucleic acid molecule encoding a polypetide a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL.

Still further aspects of the invention are a pharmaceutical composition comprising a polypetide a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL.

The polypetide a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL according to the invention represent a new stabile antigen binding portion which is useful either alone or as fusion molecules with other antibodies or antibody fragments, proteins or protein fragments.

Detailed Description of the Invention

A first aspect of the current invention is a polypetide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHl), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker are connected in one of the following orders from N-terminal to C-terminal direction: a) VH-CL-linker- VL-CHl and b) VL-CHl -linker- VH-CL; wherein VH and VL form together an antigen-binding site which binds specifically to an antigen and wherein said linker is a polypeptide of at least 30 amino acids.

Another aspect of the current invention is a mono- or multispecific antibody comprising i) two or more of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL; binding to one or more antigens; and wherein said polypeptides are fused via a peptide connector(s) with each other.

Another aspect of the current invention is a multispecific antibody comprising i) one or more of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL; binding to one or more antigens; and ii) a full length antibody specifically binding to a further antigen and consisting of two antibody heavy chains and two antibody light chains; and wherein said polypeptides under under i) are fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody. Another aspect of the current invention is said multispecifϊc antibody comprising i) one to four of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL; binding to one or more antigens; and ii) a full length antibody specifically binding to a further antigen and consisting of two antibody heavy chains and two antibody light chains; and wherein said polypeptides under under i) are fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody.

Another aspect of the current invention is said multispecific antibody comprising i) one of said polypetides a) VH-CL-linker- VL-CH 1 or b) VL-CH 1 -linker-

VH-CL; binding to one antigen; and ii) a full length antibody specifically binding to one further antigen and consisting of two antibody heavy chains and two antibody light chains; and wherein said polypeptide under under i) is fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody.

Another aspect of the current invention is said multispecific antibody comprising i) two of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CH 1-linker- VH-CL; binding to one antigen; and ii) a full length antibody specifically binding to one further antigen and consisting of two antibody heavy chains and two antibody light chains; and wherein said polypeptides under under i) are fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody.

Preferably the said multispecific antibody comprises one or two polypetides a) VH-

CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL, binding to the same antigen (bispecific antibody).

Preferably the said multispecific antibody comprises two identical polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL, binding to the same antigen (bispecific antibody).

Preferably the said multispecific antibody comprise two polypetides a) VH-CL- linker- VL-CHl or b) VL-CHl -linker- VH-CL binding to a first antigen and a second antigen (trispecific antibody). The polypeptide according to invention comprises an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHl), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: c) VH-CL-linker- VL-CHl and d) VL-CHl -linker- VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL are stabilized via the natural disulfide bond between the CL domain and the CHl domain, (see Fig. 1)

Optionally in said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker-

VH-CL also the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL) are disulfide stabilized by introduction of a disulfide bond between the following positions: i) heavy chain variable domain position 44 to light chain variable domain position

100, ii) heavy chain variable domain position 105 to light chain variable domain position 43, or iii) heavy chain variable domain position 101 to light chain variable domain position 100.

Such further disulfide stabilization of polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL is achieved by the introduction of a disulfide bond between the variable domains VH and VL of said polypeptide. Techniques to introduce unnatural disulfide bridges for stabilization for a single chain Fv are described e.g. in WO 94/029350, Rajagopal, V., et al., Prot. Engin. 10 (1997) 1453-

59; Kobayashi, H., et al., Nuclear Medicine & Biology 25 (1998) 387-393; or Schmidt, M., et al., Oncogene 18 (1999) 1711 -1721.

In one embodiment of the polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL, the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the invention is independently for each polypetide a) VH-CL-linker- VL-CHl or b) VL-CH 1-linker-

VH-CL selected from: i) heavy chain variable domain position 44 to light chain variable domain position

100, ii) heavy chain variable domain position 105 to light chain variable domain position 43, or iii) heavy chain variable domain position 101 to light chain variable domain position 100.

In one embodiment the optional disulfide bond between the variable domains is between heavy chain variable domain position 44 and light chain variable domain position 100.

In one embodiment the optional disulfide bond between the variable domains is between heavy chain variable domain position 105 and light chain variable domain position 43.

In an embodiment the polypetides a) VH-CL-linker- VL-CHl or b) VL-CH 1-linker- VH-CL without disulfide stabilization are preferred.

The term "full length antibody" denotes an antibody consisting of two "full length antibody heavy chains" and two "full length antibody light chains" (see Fig. 2). A "full length antibody heavy chain" is a polypeptide consisting in N-terminal to

C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CHl), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CHl -HR-CH2-CH3; and optionally an antibody heavy chain constant domain 4 (CH4) in case of an antibody of the subclass IgE. Preferably the "full length antibody heavy chain" is a polypeptide consisting in N-terminal to C-terminal direction of VH, CHl, HR, CH2 and CH3. A "full length antibody light chain" is a polypeptide consisting in N-terminal to C-terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL. The antibody light chain constant domain (CL) can be K (kappa) or λ (lambda). The two full length antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CHl domain and between the hinge regions of the full length antibody heavy chains. Examples of typical full length antibodies are natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD, and IgE. The full length antibodies according to the invention can be from a single species e.g. human, or they can be chimerized or humanized antibodies. The full lenght antibodies according to the invention comprise two antigen binding sites each formed by a pair of VH and VL, which both specifically bind to the same antigen. The C-terminus of the heavy or light chain of said full length antibody denotes the last amino acid at the C-terminus of said heavy or light chain. The N-terminus of the heavy or light chain of said full length antibody denotes the last amino acid at the N- terminus of said heavy or light chain.

The terms "binding site" or "antigen-binding site" as used herein denotes the region(s) of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH- CL according to the invention to which the respective antigen actually specifically binds. The antigen binding sites either in the full length antibody or in the single chain Fab fragment are formed each by a pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). An antigen-binding site of an polypetide a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL of the invention contains six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for antigen. There are three heavy chain variable domain CDRs (CDRHl, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRLl, CDRL2 and

CDRL3). The extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences.

Antibody specificity refers to selective recognition of the polypetide a) VH-CL- linker- VL-CHl or b) VL-CHl -linker- VH-CL for a particular epitope of an antigen.

Natural antibodies, for example, are monospecific. The term "multispecific" antibody as used herein denotes an antibody that has two or more antigen-binding sites of which at least two bind to a different antigen or a different epitope of the same antigen. "Bispecifϊc antibodies" according to the invention are antibodies which have two different antigen-binding specificities. Where an antibody has more than one specificity, the recognized epitopes may be associated with a single antigen or with more than one antigen. Antibodies of the present invention are e.g. multispecific for at least two different antigens, e.g. EGFR as first antigen and IGF-

IR as second antigen. In one embodiment of the invention the multispecific antibody according to the invention is bispecifϊc. In another embodiment of the invention the multispecific antibody according to the invention is trispecific.

One embodiment of the invention is a multispecific antibody according to the invention, wherein two identical polypetides a) VH-CL-linker- VL-CHl or b)

VL-CHl -linker- VH-CL binding to a second antigen are fused with their N-termini to a full length antibody via a peptide connector at the two C-termini of the two heavy chains or at the two C-termini of the two light chains of said full length antibody (tetravalent, bispecific antibody). In a preferred embodiment said multispecific antibody (preferably said tetravalent, bispecific antibody) according to the invention is containing a full length IgG and two identical said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL as described above and specifically binds human IGF-IR as well as to human EGFR. These molecules are preferably based on the antigen-binding sites of the human anti-IGF-lR antibodies <IGF-1R> HUMAB Clone 18 (DSM ACC 2587; WO 2005/005635, abbreviated as <IGF-lR>Clonel8 or <IGF-1R> AK18) and humanized <EGFR>ICR62 (WO 2006/082515 abbreviated as <EGFR>ICR62). These molecules simultaneously target and interfere with the action of two receptor tyrosine kinases on tumor cells. This dual activity causes a markedly improved anti-tumor activity compared to antibodies which interfere only with one of these receptors.

Thus in one embodiment such a multispecific antibody according to the invention is characterized in that

i) said full length antibody is specifically binding to IGFlR and comprises in the heavy chain variable domain a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDRl region of SEQ ID NO:3, and in the light chain variable domain a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a CDRl region of SEQ ID NO:6; and

ii) said polypetides a) VH-CL-linker-VL-CHl or b) VL-CHl -linker- VH-CL are specifically binding to EGFR and comprises in the heavy chain variable domain a CDR3 region of SEQ ID NO: 9, a CDR2 region of, SEQ ID NO: 10, and a CDRl region of SEQ ID NO: 11 , and in the light chain variable domain a CDR3 region of SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13, and a CDRl region of SEQ ID NO: 14.

Thus in one embodiment such a multispecific antibody according to the invention is characterized in that

i) said full length antibody is specifically binding to IGF-IR and comprises as heavy chain variable domain SEQ ID NO: 7, and as light chain variable domain SEQ ID NO: 8, and ii) said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL are specifically binding to EGFR and comprises as heavy chain variable domain SEQ ID NO: 15, and as light chain variable domain a SEQ ID NO: 16.

Thus in one embodiment such a multispecific antibody according to the invention is characterized in that

i) said full length antibody is specifically binding to EGFR and comprises in the heavy chain variable domain a CDR3 region of SEQ ID NO: 9, a CDR2 region of, SEQ ID NO: 10, and a CDRl region of SEQ ID NO: 11, and in the light chain variable domain a CDR3 region of SEQ ID NO:

12, a CDR2 region of SEQ ID NO: 13, and a CDRl region of SEQ ID NO: 14; and

ii) said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL are specifically binding to IGF-IR and comprises in the heavy chain variable domain a CDR3 region of SEQ ID NO: 1, a CDR2 region of

SEQ ID NO: 2, and a CDRl region of SEQ ID NO:3, and in the light chain variable domain a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a CDRl region of SEQ ID NO:6.

Thus in one embodiment such a multispecific antibody according to the invention is characterized in that

i) said full length antibody is specifically binding to EGFR and comprises as heavy chain variable domain SEQ ID NO: 15, and as light chain variable domain a SEQ ID NO: 16, and

ii) said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL are specifically binding to IGFlR and comprises as heavy chain variable domain SEQ ID NO: 7, and as light chain variable domain SEQ ID NO: 8.

The term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

The term "valent" as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule. A natural antibody for example or a full lenght antibody according to the invention has two binding sites and is bivalent. As such, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" denote the presence of two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antibody molecule. The multispecific antibodies according to the invention are at least

"trivalent" and may be "tetravalent", "pentavalent" or "hexavalent", preferably they are "trivalent" or "tetravalent".

Antibodies of the present invention have one or more binding sites and are mono- or multispecific, preferably bispecific or trispecific. For an antibody with more than two antigen binding sites, some binding sites may be identical, so long as the protein has binding sites for two different antigens.

The full length antibodies of the invention comprise immunoglobulin constant regions of one or more immunoglobulin classes. Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE isotypes and, in the case of IgG and IgA, their subtypes. In a preferred embodiment, an full length antibody of the invention has a constant domain structure of an IgG type antibody.

The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

The term "chimeric antibody" refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to CIq binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies.". Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g., Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.

The term "humanized antibody" refers to antibodies in which the framework: or "complementarity determining regions" (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody." See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to CIq binding and/or Fc receptor (FcR) binding.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90

(1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, GJ. MoI. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. MoI. Biol. 222 (1991) 581- 597). The techniques of Cole, et al. and Boerner, P., et al. are also available for the preparation of human monoclonal antibodies (Cole, S. P. C, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985) 77; and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention the term "human antibody" as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to CIq binding and/or FcR binding, e.g. by "class switching" i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation.)

If the polypeptides according to the invention are fused to a full lenght antibody heterodimeric fusion peptides can result, hi such case, the CH3 domains of said full lenght antibody according to the invention can be altered by the "knob-into-holes" technology which is described in detail with several examples in e.g. WO 96/02701 1, Ridgway, J.B., et al., Protein Eng 9 (1996) 617-621 ; and Merchant, A.M., et al., Nat Biotechnol 16 (1998) 677-681. In this method the interaction surfaces of the two CH3 domains are altered to increase the heterodimerisation of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole". The introduction of a disulfide bridge stabilizes the heterodimers (Merchant, A.M, et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al., J. MoI. Biol. 270 (1997) 26-35) and increases the yield. Preferred examples are e.g. a T366W mutation in the "knobs chain" and T366S, L368A, Y407V mutations in the "hole chain". But also other knobs-in-holes technologies such as the introduction of an additional disulfide bridge into the CH3 domain e.g. Y349C into the "knobs chain" and E356C or S354C into the "hole chain" and/or combined with the use of residues R409D; K370E for knobs residues and D399K;

E357K for hole residues described by EP 1870459A1, can be used.

The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

The "variable domain" (variable domain of a light chain (VL), variable region of a heavy chain (VH) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen. The domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β-sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.

The terms "hypervariable region" or "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chains of an antibody comprise from N- to C-terminus the domains FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding. CDR and FR regions are determined according to the standard definition of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

As used herein, the term "binding" or "specifically binding" refers to the binding of the antibody to an epitope of the antigen in an in vitro assay, preferably in an plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) with purified wild-type antigen. The affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), kD (dissociation constant), and KD (ko/ka). Binding or specifically binding means a binding affinity (KD) of 10"8 mol/1 or less, preferably 10"9 M to 10"13 mol/1. Thus, a polypetide a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL according to the invention is specifically binding to each antigen for which it is specific with a binding affinity (KD) of 10"8 mol/1 or less, preferably 10"9 M to 10"13 mol/1.

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.

In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

The term "linker" as used within the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin. These peptides according to invention are used to link a) VH-CL to VL-CHl or b) VL-CHl to VH-CL to form the following polypeptides according to the invention a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL. Said linker within the polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL is a peptide with an amino acid sequence with a length of at least 30 amino acids, preferably with a length of 32 to 50 amino acids. In one embodiment said linker is (GxS)n or (GxS)nGm with G = glycine, S = serine, (x =3, n= 8, 9 or 10 and m= 0, 1, 2 or 3) or (x = 4 and n= 6, 7 or 8 and m=

0, 1, 2 or 3), preferably with x = 4, n= 6 or 7 and m= 0, 1, 2 or 3, more preferably with x = 4, n= 7 and m= 2. In one embodiment said linker is (G4S)6G2.

The term "peptide connector" as used within the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin. These peptide connectors according to invention are used to fuse the single chain Fab fragments to the C-or N-terminus of the full length antibody to form a multispecific antibody according to the invention. Preferably said peptide connectors under b) are peptides with an amino acid sequence with a length of at least 5 amino acids, preferably with a length of 5 to 100, more preferably of 10 to 50 amino acids. In one embodiment said peptide connector is (GxS)n or (GxS)nGm with G = glycine,

S = serine, and (x = 3, n= 3, 4, 5 or 6, and m= 0, 1, 2 or 3) or (x = 4,n= 2, 3, 4 or 5 and m= 0, 1, 2 or 3), preferably x = 4 and n= 2 or 3, more preferably with x = 4, n= 2. In one embodiment said peptide connector is (G4S)2.

In a further embodiment said multispecific antibody according to the invention is characterized in that said full length antibody is of human IgGl subclass, or of human IgGl subclass with the mutations L234A and L235A. In a further embodiment said multispecific antibody according to the invention is characterized in that said monospecific bivalent antibody is of human IgG2 subclass.

In a further embodiment said multispecific antibody according to the invention is characterized in that said monospecific bivalent antibody is of human IgG3 subclass.

In a further embodiment said multispecific antibody according to the invention is characterized in that said full length antibody is of human IgG4 subclass or, of human IgG4 subclass with the additional mutation S228P.

Preferably said multispecific antibody according to the invention is characterized in that said full length antibody is of human IgGl subclass, of human IgG4 subclass with the additional mutation S228P.

It has now been found that the polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL or multispecific antibodies comprising such polypeptides have valuable characteristics such as biological or pharmacological activity, pharmacokinetic properties or toxicity. They can be used e.g. for the treatment of diseases such as cancer.

The term "constant region" as used within the current applications denotes the sum of the domains of an antibody other than the variable region. The constant region is not involved directly in binding of an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses, such as IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The light chain constant regions (CL) which can be found in all five antibody classes are called K (kappa) and λ (lambda).

The term "constant region derived from human origin" as used in the current application denotes a constant heavy chain region of a human antibody of the subclass IgGl, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or lambda region. Such constant regions are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785- 2788).

While antibodies of the IgG4 subclass show reduced Fc receptor (FcγRIIIa) binding, antibodies of other IgG subclasses show strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235,

Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln31 1, Asn434, and His435 are residues which, if altered, provide also reduced Fc receptor binding (Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-1 19; Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0 307 434).

In one embodiment an antibody according to the invention has a reduced FcR binding compared to an IgGl antibody and the full lenght parent antibody is in regard to FcR binding of IgG4 subclass or of IgGl or IgG2 subclass with a mutation in S228, L234, L235 and/or D265, and/ or contains the PVA236 mutation. In one embodiment the mutations in the full length parent antibody are S228P, L234A, L235A, L235E and/or PVA236. In another embodiment the mutations in the full length parent antibody are in IgG4 S228P and in IgGl L234A and L235A. Constant heavy chain regions shown in SEQ ID NO: 35 and 36. In one embodiment the constant heavy chain region of the full length parent antibody is of SEQ ID NO: 35 with mutations L234A and L235A. hi another embodiment the constant heavy chain region of the full length parent antibody is of SEQ ID NO: 36 with mutation S228P. hi another embodiment the constant light chain region of the full length parent antibody is a kappa light chain region of SEQ ID NO: 37 or lambda light chain region of SEQ ID NO: 34. Preferably the constant heavy chain region of the full length parent antibody is of SEQ ID NO: 35 or of SEQ ID NO: 36 with mutation S228P.

The constant region of an antibody is directly involved in ADCC (antibody- dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by binding of complement factor CIq to the constant region of most IgG antibody subclasses. Binding of CIq to an antibody is caused by defined protein-protein interactions at the so called binding site. Such constant region binding sites are known in the state of the art and described e.g. by Lukas, T.J., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J.J., MoI. Immunol. 16 (1979) 907-917; Burton, D.R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., MoI. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434. Such constant region binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).

The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to lysis of human target cells by an antibody according to the invention in the presence of effector cells. ADCC is measured preferably by the treatment of a preparation of antigen expressing cells with an antibody according to the invention in the presence of effector cells such as freshly isolated PBMC or purified effector cells from buffy coats, like monocytes or natural killer (NK) cells or a permanently growing NK cell line.

The term "complement-dependent cytotoxicity (CDC)" denotes a process initiated by binding of complement factor CIq to the Fc part of most IgG antibody subclasses. Binding of CIq to an antibody is caused by defined protein-protein interactions at the so called binding site. Such Fc part binding sites are known in the state of the art (see above). Such Fc part binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat). Antibodies of subclass IgGl, IgG2, and IgG3 usually show complement activation including CIq and C3 binding, whereas IgG4 does not activate the complement system and does not bind CIq and/or C3.

The polypeptide or antibody according to the invention is produced by recombinant means. Thus, one aspect of the current invention is a nucleic acid encoding the polypeptide according to the invention and a further aspect is a cell comprising said nucleic acid encoding an antibody according to the invention. Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity. For the expression of the polypeptides as aforementioned in a host cell, nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). General methods for recombinant production of antibodies are well-known in the state of the art and described, for example, in the review articles of Makrides, S. C, Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., MoI. Biotechnol. 16 (2000) 151-160; Werner, R.G., Drug Res. 48 (1998) 870-880.

The polypetides according to the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA.

Once isolated, the DNA may be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of the polypetides a) VH-CL-linker-

VL-CHl or b) VL-CHl -linker- VH-CL are prepared by introducing appropriate nucleotide changes into the polypeptide DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range, e.g. as described above. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification.

The term "host cell" as used in the current application denotes any kind of cellular system which can be engineered to generate the polypeptide according to the current invention. In one embodiment HEK293 cells and CHO cells are used as host cells. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

Expression in NSO cells is described by, e.g., Barnes, L.M., et al., Cytotechnology 32 (2000) 109-123; Barnes, L.M., et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by

Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.

The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.

A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

Purification of polypeptide is performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A., Appl. Biochem. Biotech. 75 (1998) 93-102).

One aspect of the invention is a pharmaceutical composition comprising a polypeptide a) VH-CL-linker- VL-CH 1 and b) VL-CH 1 -linker- VH-CL according to the invention or a fusion protein or multispecific antibody thereof . Another aspect of the invention is the use of a polypeptide a) VH-CL-linker- VL-CHl and b) VL-CHl -linker- VH-CL according to the invention or a fusion protein or multispecific antibody thereof for the manufacture of a pharmaceutical composition. A further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising a polypeptide according to the invention. In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing said polypeptide according to the present invention, formulated together with a pharmaceutical carrier.

One embodiment of the invention is the polypeptide a) VH-CL-linker- VL-CHl and b) VL-CHl -linker- VH-CL according to the invention or a fusion protein or multispecific antibody thereof for the treatment of cancer.

Another aspect of the invention is said pharmaceutical composition for the treatment of cancer.

Another aspect of the invention is the use of said polypeptide a) VH-CL-linker- VL-

CHl and b) VL-CHl -linker- VH-CL according to the invention or a fusion protein or multispecific antibody thereof for the manufacture of a medicament for the treatment of cancer.

Another aspect of the invention is method of treatment of patient suffering from cancer by administering said polypeptide a) VH-CL-linker- VL-CHl and b) VL-

CHl-linker-VH-CL according to the invention or a fusion protein or multispecific antibody thereof to a patient in the need of such treatment.

As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

The term cancer as used herein refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis rumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.

In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier preferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words

"transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

Where distinct designations are intended, it will be clear from the context.

The term "transformation" as used herein refers to process of transfer of a vectors/nucleic acid into a host cell. If cells without formidable cell wall barriers are used as host cells, transfection is carried out e.g. by the calcium phosphate precipitation method as described by Graham, F. L., and van der Eb, A. J., Virology

52 (1973) 456-467. However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, e.g. one method of transfection is calcium treatment using calcium chloride as described by Cohen, S.N., et al., PNAS. 69 (1972) 2110-21 14.

As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and/or to the process by which the transcribed mRNA (also referred to as transcript) is subsequently being translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectively referred to as gene product. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.

A "vector" is a nucleic acid molecule, in particular self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the functions as described. An "expression vector" is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An "expression system" usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Description of the Amino acid Sequences

SEQ ID NO: 1 heavy chain CDR3, <IGF-1R> HUMAB-Clone 18

SEQ ID NO: 2 heavy chain CDR2, <IGF- 1 R> HUMAB-Clone 18

SEQ ID NO: 3 heavy chain CDRl , <IGF-1R> HUMAB-Clone 18

SEQ ID NO: 4 light chain CDR3, <IGF- 1 R> HUMAB-Clone 18

SEQ ID NO: 5 light chain CDR2, <IGF-1 R> HUMAB-Clone 18 SEQ ID NO: 6 light chain CDRl, <IGF-1R> HUMAB-Clone 18

SEQ ID NO: 7 heavy chain variable domain, <IGF- 1 R> HUMAB-Clone 18

SEQ ID NO: 8 light chain variable domain, <IGF- 1 R> HUMAB-Clone 18

SEQ ID NO: 9 heavy chain CDR3, humanized <EGFR>ICR62

SEQ ID NO: 10 heavy chain CDR2, humanized <EGFR>ICR62 SEQ ID NO: 11 heavy chain CDRl , humanized <EGFR>ICR62

SEQ ID NO: 12 light chain CDR3, humanized <EGFR>ICR62

SEQ ID NO: 13 light chain CDR2, humanized <EGFR>ICR62

SEQ ID NO: 14 light chain CDRl , humanized <EGFR>ICR62

SEQ ID NO: 15 heavy chain variable domain, humanized <EGFR>ICR62-I- HHD

SEQ ID NO: 16 light chain variable domain, humanized <EGFR>ICR62 -I-

KC

SEQ ID NO: 17 human heavy chain constant region derived from IgGl

SEQ ID NO: 18 human heavy chain constant region derived from IgG4 SEQ ID NO: 19 kappa light chain constant region Descrjption of the Figures

Figure 1 Schematic structure of a polypetide a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL binding to a first antigen 1 according to the invention Figure 2 Schematic structure of a full length antibody binding with two antigen binding sites to a second antigen 2

Figure 3a-c Schematic structure of representative multispecific antibodies according to the invention comprising a full lenght antibody binding to a and one ore more polypetides a) VH-CL-linker- VL- CH 1 or b) VL-CH 1 -linker- VH-CL according to the invention.

Fig 3 a -tetravalent bispecific format with two identical VH-CL- linker- VL-CHl polypeptides (also two identical VL-CH 1-linker- VH-CL polypeptides in such a tetravalent bispecific antibody) Fig 3b -trivalent bispecific format with one VH-CL-linker- VL- CH 1 polypeptide (also two identical VL-CH 1 -linker- VH-CL polypeptides result in such a trivalent bispecific antibody) Fig 3 c -tetravalent trispecific format with one VH-CL-linker- VL- CHl polypeptide and one VL-CHl -linker- VH-CL polypeptide binding to different antigens (also two different a) VH-CL-linker- VL-CHl polypeptides binding to different antigens or two different b) VL-CHl -linker- VH-CL polypeptides binding to different antigens result in such a tetravalent trispecific antibody).

Experimental Procedure Examples

Materials & general methods

General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E., A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered and referred to according to EU numbering (Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991). Recombinant DNA techniques

Standard methods are used to manipulate DNA as described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents are used according to the manufacturer's instructions.

Gene synthesis

Desired gene segments are prepared from oligonucleotides made by chemical synthesis. The 600 - 1800 bp long gene segments, which are flanked by singular restriction endonuclease cleavage sites, are assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites e.g. BamHI/BstEII, BamHI/BsiWI, BstEII/Notl or BsiWI/Notl into a pcDNA 3.1/Zeo(+) (Invitrogen) based on a pUC cloning vector. The DNA sequences of the subcloned gene fragments are confirmed by DNA sequencing. Gene synthesis fragments are ordered according to given specifications at Geneart (Regensburg, Germany).

DNA sequence determination

DNA sequences are determined by double strand sequencing performed at Sequiserve GmbH (Vaterstetten, Germany).

DNA and protein sequence analysis and sequence data management

The GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Invitrogens Vector NTl Advance suite version 9.1 is used for sequence creation, mapping, analysis, annotation and illustration.

Cell culture techniques

Standard cell culture techniques are used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M., (eds.), John Wiley & Sons, Inc.

Transient expression of immunoglobulin variants in HEK293F cells

The polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL or multispecific antibodies thereof are expressed by transient transfection of human embryonic kidney 293-F cells using the FreeStyle™ 293 Expression System according to the manufacturer's instruction (Invitrogen, USA). Briefly, suspension FreeStyle™ 293-F cells are cultivated in FreeStyle™ 293 Expression medium at 37°C/8 % CO2 and the cells are seeded in fresh medium at a density of 1-2x106 viable cells/ml on the day of transfection. The DNA-293fectin™ complexes are prepared in Opti-MEM® I medium (Invitrogen, USA) using 333 μl of 293fectin™ (Invitrogen, Germany) and 250 μg of heavy and light chain plasmid DNA in a 1 :1 molar ratio for a 250 ml final transfection volume. Bispecific antibody containing cell culture supematants are clarified 7 days after transfection by centrifugation at 14000 g for 30 minutes and filtration through a sterile filter (0.22 μm).

Supematants are stored at -20° C until purification.

Protein determination

The protein concentration of purified antibodies and derivatives is determined by determining the optical density (OD) at 280 nm with the OD at 320nm as the background correction, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, CN. , et al., Protein Science 4 (1995) 2411-2423.

Antibody concentration determination in supematants

The concentration of antibodies and derivatives in cell culture supematants is measured by affinity HPLC chromatography. Briefly, cell culture supematants containing antibodies and derivatives that bind to Protein A are applied to an

Applied Biosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH 7.4 and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH 2,5 on an UltiMate 3000 HPLC system (Dionex). The eluted protein is quantified by UV absorbance and integration of peak areas. A purified standard

IgGl antibody serves as a standard.

Protein purification

The secreted antibodies are purified from the supernatant in two steps by affinity chromatography using Protein A-Sepharose™ (GE Healthcare, Sweden) and Superdex200 size exclusion chromatography. Briefly, the polypetide a) VH-CL- linker- VL-CHl or b) VL-CHl -linker- VH-CL comprising antibody containing clarified culture supematants are applied on a HiTrap ProteinA HP (5 ml) column equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins are washed out with equilibration buffer. The antibodies are eluted with 0.1 M citrate buffer, pH 2.8, and the protein containing fractions are neutralized with 0.1 ml 1 M Tris, pH 8.5. Then, the eluted protein fractions are pooled, concentrated with an Amicon Ultra centrifugal filter device (MWCO: 30 K, Millipore) to a volume of 3 ml and loaded on a

Superdex200 HiLoad 120 ml 16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 2OmM Histidin, 140 mM NaCl, pH 6.0. Monomelic antibody fractions are pooled, snap-frozen and stored at -80°C. Parts of the samples are provided for subsequent protein analytics and characterization.

Analysis of purified proteins

The protein concentration of purified protein samples is determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. The purity of the antibodies are analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue. The NuP AGE®

Pre-Cast gel system (Invitrogen, USA) is used according to the manufacturer's instruction (4-20 % Tris-Glycine gels). The aggregate content of antibody samples is analyzed by high-performance SEC on an UltiMate 3000 HPLC system (Dionex) using a Superdex 200 analytical size-exclusion column (GE Healthcare, Sweden) in 200 mM KH2PO4, 250 mM KCl, pH 7.0 running buffer at 25°C. 25 μg protein are injected on the column at a flow rate of 0.5 ml/min and eluted isocratic over 50 minutes. For stability analysis, concentrations of 0.1 mg/ml, 1 mg/ml and 3 mg/ml of purified proteins are prepared and incubated at 4°C, 37°C for 7 days and then evaluated by high-performance SEC. The integrity of the amino acid backbone of reduced bispecific antibody light and heavy chains is verified by NanoElectrospray

Q-TOF mass spectrometry after removal of N-gl yeans by enzymatic treatment with Peptide-N-Glycosidase F (Roche Molecular Biochemicals).

Design of polypeptide a) VH-CL-linker- VL-CHl or b) VL-CHl-linker- VH-CL comprising multispecific antibody molecules

In the following as one embodiment of the invention tetravalent bispecific antibodies comprising a full length antibody binding to a first antigen with two identical polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL binding to a second different antigen connected via a peptide connector to the full length antibody (either both polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL at the two C-termini of the heavy chain or at the two C-termini of the light chain) are exemplified.

E.g. as heavy chain variable domain VH for the two first antigen binding sites of the full lenght antibody, one of the two sequences SEQ ID NO: 15 or SEQ ID NO: 7 can be used. As light chain variable domain VL for the two first antigen binding sites of the full lenght antibody, one of the two sequences SEQ ID NO: 16 or SEQ ID NO: 8 can be used. Then, as heavy chain variable domain VH for the two second antigen binding sites of the two identical polypetides a) VH-CL-linker- VL- CHl or b) VL-CHl -linker- VH-CL, the other of the two sequences SEQ ID NO: 15 or SEQ ID NO: 7 is used. As light chain variable domain VL for the two second antigen binding sites of the two identical polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl -linker- VH-CL, the other of the two sequences SEQ ID NO: 16 or SEQ ID NO: 8 is used.

By gene synthesis and recombinant molecular biology techniques, VL-CHl and VH-CL, comprising the VH and VL of the respective antigen binding site are linked by a glycine serine (G4S)nGm single-chain-linker to give a polypetides a) VH-CL-linker- VL-CHl or b) VL-CH 1 -linker- VH-C, which is attached to the C- terminus of the antibody heavy or light chain using (G4S)n linker.

Optionally , cysteine residues are introduced in the VH (including Kabat position 44,) and VL (including Kabat position 100) domain of the polypetides a) VH-CL- linker- VL-CHl or b) VL-CHl -linker- VH-CL according to techiques as described earlier (e.g. WO 94/029350; Reiter, Y., et al., Nature biotechnology 14 (1996) 1239-1245; Young, N.M., et al., FEBS Letters 377 (1995) 135-139; or Rajagopal, V., et al., Protein Engineering 10 (1997) 1453-59).

All these molecules are recombinantly produced, purified and characterized and protein expression, stability and biological activity is evaluated.

A summary of the multispecific antibody designs that are applied to generate tetravalent, bispecific antibodies e.g. <EGFR-IGF-1R> or <IGF-1R-EGFR> antibodies is given in Table 1 and Figure 3 a. For this study, we use the term 'cross- scFab-XGFR' or 'XscFab-XGFR' to describe the various tetravalent protein entities. A general representation of different format designs is shown in Figure 3a- c. Table 1 - The different bispecific tetravalent antibody formats with C- terminal single chain Fab fragment attachments and the corresponding XscFab-XGFR- nomenclature. An "-" in the table means "not present"

Figure imgf000033_0001

Example 1

Expression & Purification of bispecific <EGFR-IGF1R> antibody

XscFabXGFRl molecules

Light and heavy chains of the corresponding bispecific antibodies are constructed in expression vectors carrying pro- and eukaryotic selection markers. These plasmids are amplified in E.coli, purified, and subsequently transfected for transient expression of recombinant proteins in HEK293F cells (utilizing Invitrogen's freesyle system). After 7 days, HEK 293 cell supernatants are harvested and purified by protein A and size exclusion chromatography. Homogeneity of all bispecific antibody constructs is confirmed by SDS-PAGE under non reducing and reducing conditions.

The bispecific antibodies are expressed by transient transfection of human embryonic kidney 293 -F cells using the FreeStyle™ 293 Expression System according to the manufacturer's instruction (Invitrogen, USA). Briefly, suspension FreeStyle™ 293 -F cells are cultivated in FreeStyle™ 293 Expression medium at 37°C/8 % CO2 and the cells are seeded in fresh medium at a density of 1-2x106 viable cells/ml on the day of transfection. The DNA-293fectin™ complexes are prepared in Opti-MEM® I medium (Invitrogen, USA) using 333 μl of 293fectin™

(Invitrogen, Germany) and 250 μg of heavy and light chain plasmid DNA in a 1 :1 molar ratio for a 250 ml final transfection volume. Bispecific antibody containing cell culture supernatants are clarified 7 days after transfection by centrifugation at 14000 g for 30 minutes and filtration through a sterile filter (0.22 μm). Supernatants are stored at -20° C until purification.

The secreted antibodies are purified from the supernatant in two steps by affinity chromatography using Protein A-Sepharose™ (GE Healthcare, Sweden) and Superdex200 size exclusion chromatography. Briefly, the bispecific antibody containing clarified culture supernatants are applied on a HiTrap ProteinA HP (5 ml) column equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins are washed out with equilibration buffer. The bispecific antibodies are eluted with 0.1 M citrate buffer, pH 2.8, and the protein containing fractions are neutralized with 0.1 ml 1 M Tris, pH 8.5. Then, the eluted protein fractions are pooled, concentrated with an Ami con Ultra centrifugal filter device (MWCO: 30 K, Millipore) to a volume of 3 ml and loaded on a Superdex200 HiLoad 120 ml 16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 2OmM Histidin, 140 mM NaCl, pH 6.0. Monomelic antibody fractions are pooled, snap-frozen and stored at -80°C. Part of the samples are provided for subsequent protein analytics and characterization.

Claims

Patent Claims
1. A polypetide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHl), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker are connected in one of the following orders from N-terminal to C-terminal direction: a) VH-CL-linker- VL-CHl and b) VL-CHl -linker- VH-CL; wherein VH and VL form together an antigen-binding site which binds specifically to an antigen, and wherein said linker is a polypeptide of at least 30 amino acids.
2. A mono- or multispecific antibody comprising i) two or more of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL according to claim 1, binding to one or more antigens; and wherein said polypeptides are fused via a peptide connector(s) with each other.
3. A multispecific antibody comprising i) one or more of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL, binding to one or more antigens; and ii) a full length antibody specifically binding to a further antigen and consisting of two antibody heavy chains and two identical antibody light chains; and wherein said polypeptides under under i) are fused to said full length antibody under a) via a peptide connector at the C- or N- terminus of the heavy or light chain of said full length antibody.
4. The multispecific antibody according to claim 3 , comprising i) one to four of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CHl- linker- VH-CL; binding to one or more antigens.
5. The multispecific antibody according to claim 3 , comprising i) one of said polypetides a) VH-CL-linker- VL-CH 1 or b) VL-CH 1 -linker-
VH-CL; binding to one antigen.
6. The multispecific antibody according to claim 3 , comprising i) two of said polypetides a) VH-CL-linker- VL-CHl or b) VL-CH 1-linker- VH-CL, binding to one antigen
7. The multispecific antibody according to any one of claims 3 to 6 , multispecific antibody comprises two identical polypetides a) VH-CL-linker-
VL-CHl or b) VL-CHl -linker- VH-CL, binding to the same antigen.
8. A pharmaceutical composition comprising an antibody according to claims 1 to 7
9. A nucleic acid encoding a polypetide according to claim 1 ccording to claims 1 to 7.
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